Anti mouse fasl pe

To visualize Fas localization to the T cell synapse, transduced T cells and FBL tumor cells were combined at a 10:1 ratio, and conjugates were formed by centrifugation (200×g), followed by incubation at 37°C for 30 min. 30 µl of cell suspension was added to an Ibidi μ-Slide VI^0.4 (80606, Ibidi) microscopy slide and incubated at 37°C for 15 min. Cell conjugates were fixed and stained in PBS following the Ibidi staining protocol with Fc Block (553142, BD PharMingen), anti-mouse Fas APC (563647, BD Biosciences), and anti-mouse FasL PE (106805, Biolegend).
To determine mitochondria concentration, MitoTracker Deep Red (M22426, Invitrogen) was resuspended in serum-free RPMI (11875–093, Gibco) to a final concentration of 10 nM (1:100,000) and warmed to 37°C before cells were resuspended in the solution at 10⁶ cells/ml and incubated at 37°C for 30 min. Cells were resuspended in PBS and stained in Ibidi μ-Slide VI^0.4 (80606, Ibidi) as described above, with Fc Block (553142, BD PharMingen), Hoechst (33342, ThermoFisher), and cholera toxin subunit B AF594 (C34777, Life Technologies). All fluorescent microscopy was performed using a DeltaVision Elite high-resolution microscope (GE Healthcare Life Sciences). Captured images were analyzed with ImageJ (National Institutes of Health).